Complement dependent cytotoxicity activity of therapeutic antibody fragments is acquired by immunogenic glycan coupling

Oligosaccharides are implicated in the development of the immune response notably in complement activation. Anti-tumoural immunotherapy by monoclonal antibodies (mAbs) offers some advantages to chemotherapy including cell targeting but some of them are inefficient to generate cytotoxicity dependent complement (CDC) known to be important in the antibody’s efficacy. The aim of this study is to give a CDC activity of mAb by linkage of a complement activating oligosaccharide to this antibody via a hetero-bifunctional linker allowing control of the conjugation reaction. We worked on non Hodgkin Burkitt’s lymphoma as cancer source, Fab fragments of rituximab devoid of complement activity as mAb and the trisaccharide Gal alpha(1→3)Gal beta(1→4)GlcNAc as immunogenic glycan. The bioconjugate Fab-Gal was characterized by biochemical methods and we demonstrated that the α-Gal epitope was recognized by seric immunoglobulins. After checking the recognition capacity of the Fab-Gal conjugate for the CD20 epitope, in vitro assays were performed to evaluate the activation of the complement cascade by the Fab-Gal conjugate. The effect of this bioconjugate was confirmed by the evaluation of the proliferation response of Burkitt’s cell line. The relative facility realization of this strategy represents new approaches to increase activities of mAbs.

Imunizações pré-clínicas contra malária baseadas no Antígeno 1 de Membrana Apical (PvAMA-1): testes de protocolos homólogos e heterólogos de indução e reforço utilizando DNA plasmidial e/ou proteína recombinante / Pre-clinical immunizations against malaria based on the Apical Membrane Antigen 1 (PvAMA-1): testing prime-boost homologous and heterologous protocols using DNA and/or recombinant protein

O Antígeno 1 de Membrana Apical (AMA-1) é um dos mais promissores candidatos a vacina contra a fase sanguínea da malária. No presente estudo, geramos uma nova proteína recombinante baseada no ectodomínio de AMA-1 de P. vivax (PvAMA-1) a partir do gene sintético com códon otimizado para expressão secretada na levedura Pichia pastoris. Por ELISA, a proteína PvAMA-1 foi reconhecida por 62,5% dos soros de pacientes da Amazônia brasileira infectados por P. vivax. A imunogenicidade de PvAMA-1 foi avaliada em camundongos BALB/c, utilizando protocolos homólogos e heterólogos de indução e reforço com DNA plasmidial e/ou proteína recombinante, emulsificada em Adjuvante de Freund. Os resultados mostraram que a eficiência da imunização é dependente da presença da proteína emulsificada em adjuvante forte. As imunizações subseqüentes foram realizadas com a proteína recombinante emulsificada em diferentes adjuvantes: sais de alumínio (Alum), Monophosphoryl Lipid A (MPLA) de Bordetella pertussis, flagelina FliC de Salmonella Typhimurium, saponina Quil A e Adjuvante Incompleto de Freund (AIF). Os resultados demonstraram que as imunizações na presença de Quil A e AIF foram capazes de induzir altos títulos de anticorpos IgG e uma resposta de isotipos de IgG mais balanceada, caracterizando um padrão do tipo Th1/Th2. Títulos de anticorpos mais baixos, mas também expressivos, foram obtidos na presença de Alum, MPLA, Alum + MPLA e Alum + FliC. A análise da resposta proliferativa, por citometria de fluxo utilizando marcação com CFSE, mostrou que células esplênicas CD3+CD4+, assim como CD3+CD8+, de animais imunizados com PvAMA-1 na presença dos adjuvantes Alum, Alum + MPLA, Quil A e AIF foram capazes de proliferar especificamente in vitro. Por imunofluorescência, soros policlonais de camundongos imunizados com o antígeno na presença de MPLA e Quil A foram capazes de reconhecer a proteína nativa expressa em isolados de P. Vivax da Ásia. Visando futuros testes pré clínicos em primatas não humanos, a proteína PvAMA-1 foi produzida em boas práticas de laboratório (BPL) por uma companhia especializada e o potencial imunogênico da proteína foi confirmado em imunizações posteriores. Em conjunto, nossos resultados demonstram que PvAMA-1 é um antígeno promissor para ser explorado em protocolos de imunização contra malária vivax, individualmente, ou em combinação com outros antígenos

The Apical Membrane Antigen 1 (AMA-1) is one of the most promissing vaccine candidates against the erythrocytic stage of malaria. In the present study we generated a new recombinant protein based on AMA-1 ectodomain of P. vivax (PvAMA-1) using a synthetic codon optimized gene for yeast secreted expression. By ELISA, the protein PvAMA-1 was recognized by 62,5% of the sera from Brazilian Amazonia patients infected by P. vivax. The immunogenicity of PvAMA-1 was evaluated in BALB/c mice using homologous and heterologous protocols of prime and boost with plasmidial DNA and/or recombinant protein emulsified in Freund adjuvant. The results showed that the efficiency of immunization is dependent on the presence of a strong adjuvant. The following immunizations were conducted using the protein emulsified in different adjuvants: aluminium salts (Alum), Monophosphoryl Lipid A (MPLA) of Bordetella pertussis, flagelin FliC of Salmonella Typhimurium, saponin Quil A and Incomplete Freund's Adjuvant (IFA). The results showed that immunizations in the presence of Quil A e IFA were able to induce high IgG titers and a more balanced IgG isotypes response, featuring a standard type Th1/Th2. Lower antibody titers, but also significant, were obtained in the presence of Alum, MPLA, Alum + MPLA and Alum + FliC. The proliferative cellular analysis, by flow cytometry using CFSE staining, showed that splenic cells CD3+CD4+, as well as CD3+CD8+ from immunized mice that received PvAMA-1 with Alum, Alum + MPLA, Quil A and IFA were specifically able to proliferate in vitro. By immunofluorescence, the polyclonal sera from mice immunized with the antigen in the presence of MPLA and Quil A were able to recognize native protein expressed in Asian P. vivax isolates. Aiming future pre-clinical assays in non-human primates, the protein PvAMA-1 was produced in good laboratory practices (GLP) conditions by a specialized company and its immunogenic efficiency was confirmed in later immunizations. Altogether, our results showed that PvAMA-1 is a promissor antigen to be explored in immunization protocols against vivax malaria, individually, or combined with other antigens.

Removal of αGal xenotransplantation antigen by a novel α-galactosidase / 中国实验血液学杂志

αGal, a xenotransplantations antigen (XTA), can lead to hyper acute reaction (HAR) in xenotransplantation. α-Galactosidase from B. fragilis is a novel galactosidase belong to CAZy GH110 which can clear the terminal αGal from branched and linear oligosaccharides. This study was purposed to investigate the removal effect of a novel α-galactosidase on α-Gal XTA on surface of red blood cells. The αGal XTA from the red blood cells of cattle, pig, dog and rabbit was digested by using recombinant α-galactosidase; the α-Gal antigens on surface of cells was detected by flow cytometry. The results showed that the XTA was disappeared completely or mainly. It is concluded that the novel α-galactosidase is a potential enzyme to remove the XTA on the surface of xenotransplants and can be used to overcome the HAR in xenotransplantation.

Effect of sequential intratumoral injection of xenogeneic antigens for immunotherapy in immunized mice bearing S180 tumor / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the therapeutic effect of sequential intratumoral injection of xenogeneic antigens in immunized tumor-bearing mice.</p><p><b>METHODS</b>Sequential intratumoral injection of the xenoantigens was performed in immunized mice bearing S180 tumor. The tumor size changes were observed, and the tumor-infiltrating lymphocytes (TIL) including CD3+CD4+T, CD3+CD8+T, and CD3+CD4+CD25+T lymphocytes were counted with flow cytometry. The concentrations of IL-2 and TNF-alpha in the tumor was measured using ELISA.</p><p><b>RESULTS</b>No significant difference was found in the number of CD3+T lymphocytes in the TILs between different groups. After the immunotherapy, the percentages of CD3+CD4+T, CD3+CD8+T and CD3+CD4+CD25+T lymphocytes were 54%, 22% and 2.91%, respectively, with the CD4+/CD8+ ratio of 2.49, significantly different from that in the control group (P<0.05). The concentrations of IL-2 and TNF-alpha were 100.61 pg/ml and 54.114 pg/ml, respectively, significantly different from those in the control group (P<0.05).</p><p><b>CONCLUSION</b>Sequential intratumoral injection of heteragenetic antigena can significantly increase the amount of effector cells and cytokines in the micro-environment of the tumor, and decrease the expression of T regulatory.</p>

Avaliação histológica do implante do osso fetal bovino acelular e desmineralizado em subcutâneo de ratos / Histological evaluation of acellular and demineralized fetal bovine bone in the subcutaneous of rats

Objetivo: Avaliar a resposta tecidual ao implante do osso fetal bovino (FBB) acelular e desmineralizado em subcutâneo de ratos. Métodos: O FBB foi obtido em frigorífico local, transportado em gelo para o laboratório, onde foi lavado, tratado mecânica e quimicamente (álcalis e solventes orgânicos) para remoção de débris, células e sangue; a desmineralização do material foi obtida com ácido clorídrico 0,6M a 4°C e neutralização com tampão fosfato em pH 7,4). Foram utilizados 40 ratos machos Wistar divididos em dois grupos: teste e controle (n = 5). No grupo teste um bloco de FBB (1 x 1 x 1cm) foi implantado no tecido subcutâneo de cada animal. No grupo controle os procedimentos cirúrgicos foram idênticos aos do grupo teste, mas sem a colocação do material. Após 10, 20, 30 e 60 dias os animais foram mortos e as peças coletadas, fixadas em formol a 10 por cento tamponado, lavadas, desidratadas, diafanizadas em xilol e incluídas em parafina. Cortes com 6µm de espessura foram corados com hematoxilina-cosina. Resultado: A análise microscópica indicou que o osso fetal bovino foi circundado por tecido conjuntivo aos 10 dias. Entre 20 e 30 dias observou-se a substituição gradual do tecido conjuntivo em contato com o material implantado por tecido adiposo. No período de 60 dias notou-se acentuada diminuição das trabéculas ósseas do material implantado, sugerindo discreta absorção do material implantado e a substituição do tecido conjuntivo frouxo que circundava as partículas por tecido adiposo. Em nenhum dos períodos experimentais foram observados linfócitos ou plasmócitos no tecido reacional ou destruição tecidual. Raras áreas apresentaram células gigantes multinucleadas. A análise semiquantitativa das lâminas não demonstrou diferença significativa (p>0,05) entre os grupos controle e FBB com relação ao infiltrado inflamatório, presença de vasos e grau de fibrosamento. Conclusão: Considerando que o material não induziu destruição tecidual ou recrutamento de células do sistema imunológico, foi parcialmente absorvido e bem tolerado pelo tecido, os autores concluem que o osso fetal bovino foi biocompatível

Antigen measurement and biomechanical characteristics of the inbred-line Banna mini-pig acellular bone matrix / 生物医学工程学杂志

In this study, we prepared the acellular bone matrix of the inbred-line Banna mini-pig by using tissue engineering method and evaluated its possible application in bone tissue engineering. Histological analysis, xenoantigen expression and biomechanical measurement were performed on the matrix. HE staining and scanning electron microscopy showed the cellular components were almost removed. Immunohischemical result demonstrated that the xenoantigen, alpha-gal,was also eliminated. There was no statistically significant difference between the acellular bone matrix group and control group. The acellular bone matrix can provide appropriate space structure and strength for grafts. In conclusion, our data suggest that acellular bone matrix is a new kind of ideal bone scaffold material.

Bacillus subtilis as a tool for vaccine development: from antigen factories to delivery vectors

Bacillus subtilis e alguns de seus parentes mais próximos possuem uma longa história de aplicações industriais e biotecnológicas. A busca de sistemas de expressão de antígenos baseados em linhagens recombinants de B. subtilis mostra-se atrativa em função do conhecimento genético disponível e ausência de uma membrana externa, o que simplifica a secreção e a purificação de proteínas heterólogas. Mais recentemente, esporos geneticamente modificados de B. subtilis foram descritos com veículos indestrutíveis para o transporte de antígenos vacinais. Todavia a produção e o transporte de antígenos por linhagens de B. subtilis encontra obstáculos, como a expressão gênica instável e imunogenicidade reduzida, que podem ser superados com o auxílio de tecnologias genéticas atualmente disponíveis. Apresentamos nesta revisão o estado atual da pesquisa em vacinas baseadas em B. subtilis, empregado tanto como fábrica de proteínas ou veículos, e discute algumas alternativas para o uso mais adequado de linhagens geneticamente modificadas.

Comparison of modification of surface xenoantigens on bovine and porcine erythrocytes / 中国实验血液学杂志

This study was aimed to explore impact of removal of cell membrane G alalpha1-3Gal beta1-4Glc NAc epitopes (called alpha-Gal) and chemical modification of other xenoantigen on bovine red blood cell (bRBC) and porcine red blood cell (pRBC) antigenicity and to compare their modified erythrocytes, in order to provide basis for development of human blood substitute with rich source, high safety and efficacy. bRBC and pRBC were subjected to both enzymatic removal of membrane alpha-Gal with recombinant coffee bean alpha-galactosidase (rC alpha-GalE) and covalent attachment of benzotriazole carbonate-linked methoxypolyethylene glycol (mPEG-BTC, MW = 20 kD). The effects of treatment were measured by hemagglutination, flow cytometric assay of IgG binding and clinical cross-match testing to human sera. The results showed that although alpha-galactosidase treatment reduced hemagglutination titers to levels similar to negative control, the combination of the treatments was most effective. Clinically used cross-match tests between bRBC, pRBC and human sera demonstrated increased compatibility. Bovine RBC were more robust than pRBC, and had less xenoantigens, and had longer half life than pRBC in vivo. These characteristics suggested that bRBCs were more suitable to investigation as an alternatives to hRBC in clinical transfusion than pRBC. These data suggested that strategies to remove or mask xenoantigens on bRBC reduce antigenicity sufficiently to allow in vitro cross-match compatibility to human sera, and therefore bRBC following modification may be considered as human blood substitute.

Rat hippocampus and primary immune response.

During immune challenge hippocampal region shows time-dependent changes in neurotransmitter levels. Hence in the present study the effect of electrolytic lesion in the dorsolateral hippocampus (DLH) and ventral hippocampal formation (VHF) (to create a disturbance in neurotransmitter levels) on humoral immunity in albino rats has been studied along with appropriate controls. Haemagglutination titre, IgM and IgG levels were monitored on the 5th day after an immune challenge by sheep red blood cells (SRBC) suspension. Antigen challenged lesioned animals had low circulating antibody titre levels compared with the controls and their site-specific sham lesioned groups. The IgM levels were significantly lowered in both DLH and VHF lesioned and immunized animals compared to their immunized sham groups as well as immunized controls. However, only immunized VHF lesioned group showed a significant decrease in IgG level from their immunized sham group. It was concluded from the results that an intact hippocampal region is essential for the normal humoral immunity for the primary immune response in rats. Probably VHF region may be required for the secondary immune response as indicated by the alteration in IgG levels in these animals.

Salmonella como vetor de vacinas vivas orais / Live oral vaccines using salmonella

Hß alguns anos, foram desenvolvidas as primeiras linhagens atenuadas de Salmonella para serem utilizadas como candidatas a vacinas vivas orais contra a febre tifóide. No início, ainda eram desconhecidas as mutações responsáveis pelo fenótipo atenuado, mas, com o acúmulo de conhecimento sobre a genética associada à virulência, surgiram novas linhagens com atenuações geneticamente definidas. Muitas linhagens de S. enterica sorotipo Typhimurium e S. enterica sorotipo Typhi já foram bem estudadas quanto à capacidade de induzir resposta imunológica em modelos animais e em humanos. Com o desenvolvimento de sistemas de clonagem e expressão eficientes, o uso destas linhagens vacinais extrapolou o problema das salmoneloses, uma vez que tornou-se possível a produção e administração de antígenos de diferentes agentes patogênicos. Recentemente, uma nova tecnologia que vem sendo explorada é o uso de Salmonella como carreadora de vacinas de DNA. Tais vacinas já se mostraram capazes de induzir potentes respostas humorais e celulares contra antígenos heterólogos nos organismos hospedeiros. Todo este progresso nos estudos com as linhagens vacinais de Salmonella demonstra o potencial que elas possuem para a produção das futuras vacinas contra doenças infecciosas, parasitárias e até mesmo contra o câncer.

Expression of human alpha-galactosidase and alpha1,2-fucosyltransferase genes modifies the cell surface Galalpha1,3Gal antigen and confers resistance to human serum-mediated cytolysis / 中国医学科学杂志(英文版)

<p><b>OBJECTIVE</b>To explore the strategies which reduce the amount of xenoantigen Galalpha1,3Gal.</p><p><b>METHODS</b>Human alpha-galactosidase gene and alpha1,2-fucosyltransferase gene were transferred into cultured porcine vascular endothelial cells PEDSV.15 and human alpha-galactosidase transgenic mice were produced. The Galalpha1,3Gal on the cell surface and susceptibility of cells to human antibody-mediated lysis were analyzed.</p><p><b>RESULTS</b>Human alpha-galactosidase gene alone reduced 78% of Galalpha1,3Gal on PEDSV.15 cell surface while human alpha-galactosidase combined with alpha1,2-fucosyltransferase genes removed Galalpha1,3Gal completely. Decrease of Galalpha1,3Gal could reduce susceptibility of cells to human antibody-mediated lysis, especially during co-expression of alpha-galactosidase gene and alpha1,2-fucosyltransferase gene. RT-PCR indicated positive human alpha-galactosidase gene expression in all organs of positive human alpha-galactosidase transgenic F1 mice including heart, liver, kidney, lung, and spleen, the amount of Galalpha1,3Gal antigens on which was reduced largely. 58% of spleen cells from F1 mice were destroyed by complement-mediated lysis compared with 24% of those from normal mice.</p><p><b>CONCLUSIONS</b>Human alpha-galactosidase gene and alpha1,2-fucosyltransferase gene effectively reduce the expression of Galalpha1,3Gal antigens on endothelial cell surface and confers resistance to human serum-mediated cytolysis. The expression of human alpha-galactosidase in mice can also eliminate the Galalpha1,3Gal antigens in most tissues and decrease the susceptibility of spleen cells to human serum-mediated cytolysis.</p>

Heterologous antigen extract in ELISA for the detection of human IgE anti-Strongyloides stercoralis

Extrato contendo larvas de Strongyloides ratti foi usado na padronização de um ELISA para detecção de IgE gênero-específica na estrongiloidíase humana. Foram analisadas 40 amostras de soro de pacientes monoinfectados que estavam eliminando larvas de S. stercoralis nas fezes (Grupo I), 40 de pacientes com outros parasitos intestinais (Grupo II), e 40 indivíduos copronegativos (Grupo III). Níveis de IgE gênero-específica (índice ELISA: EI) foram significativamente maiores no Grupo I (EI = 1,43) do que no II (EI = 0,70) e III (EI = 0,71), mostrando positividade de 55 por cento, 2,5 por cento e 0 por cento, respectivamente. Similarmente, soros dos pacientes copropositivos (Grupo I) apresentaram níveis significativamente maiores de IgE total (866 IU/mL) quando comparados com os soros dos Grupo II (302 IU/mL) e III (143 IU/mL). Uma significativa correlação positiva foi encontrada entre os níveis de IgE específica a Strongyloides sp. e IgE total nos soros de pacientes com estrongiloidíase. Em conclusão, extrato heterólogo de S. ratti mostrou ser uma ferramenta útil para detecção de IgE gênero-específica por ELISA, desta forma contribuindo para melhor caracterização do perfil da resposta imune na estrongiloidíase humana.

Expressão de antígenos de Bordetella pertussis em BCG recombinante: subunidade 1 da toxina pertussis e fragmento A da hemaglutinina filamentosa (FHA) / Expression of Bordetella pertussis antigens in recombinant BCG: 1 subunit of pertussis toxin and fragment A of Filamentous hemagglutinin (FHA)

O desenvolvimento de vacinas multivalente constitui uma das prioridades na pesquisa de vacinas modernas. A utilização de vetores vivos para apresentação de antígenos heterólogos mostra-se bastante atraente, e eliminaria a necessidade de várias doses para se alcançar uma proteção máxima. Este tipo de vacina poderia ser administrado em dose única, o que poderia aumentar a cobertura vacinal. Neste trabalho foi explorado o potencial do Mycobacterium bovis BCG recombinante (rBCG) vivo expressando antígenos de Bordetella pertussis, como futuro componente de uma vacina tetravalente rBCG-DTP contra a tuberculose, tétano, difteria e pertussis. Os antígenos de pertussis utilizados foram a subunidade S1 mutada e atóxica da Toxina Pertussis (S1-PT) e o fragmento CRD, um domínio imunogênico da proteína FHA (Adesina Hemaglutinina Filamentosa)...

Antigen expression and the biomechanical characteristics of the biologic blood vessel matrix / 生物医学工程学杂志

To explore the changes of the antigen expression and the biomechanical characteristics of blood vessel in Banna little ear pig before and after trypsin treatment, and provide data for xenotransplantation and pig vessel using for tissue engineering. Geometric morphology and microstructure of pig cartoid artery were stuided quantitatively by histologic method and computer image analysis. The relationship between pressure and diameter was observed at different period of time before and after trypsin treatment. Affinity-immunohistochemistry assay was conducted to detect the expression of xenoantigens (alpha-Gal). The results showed that alpha-Gal antigen is only expressed in vascular endothelial cellsouly. There is no significant difference in blood vessel compliance. These demonstrate that the antigenicity of pig carotid artery is significantly reduced, however, the mechanical characteristics did not change significantly. We suppose that pig vessels treated by trypsin can be used as the substrate material for vascular tissue engineering.

Vacuna atenuada de Salmonella como vector de antígenos heterólogos / Attenuated Salmonella vaccine as a vector for heterologous antigens

Salmonella enterica, serotipo Thphy, es el agente de la fiebre tifoidea de los habitantes de las regiones más pobres del mundo y es, además, el centro de atención de muchos investigadores dados sus fascinantes mecanismos de invasion, multiplicación intracelular y diseminación intercelular que expresa in vivo e in vitro. Aunque estos mecanismos se asocian directamente con la patogenicidad y la severidad de la enfermedad, las mutaciones definidad en el cromosoma de Salmonella han permitido que estos mecanismos de virulencia se puedan utilizar en beneficio del hopedero. Las mutantes atenuadas de Salmonella son capaces de invadir las células M de la mucosa intestinal y de migrar a las células linfoides del sistema reticuloendotelial donde, en lugar de causar enfermedad, activan eficazmente las respuesta inmunes humoral y celular no sólo contra el microorganismo mismo sino también contra aquellos antígenos heterólogos recombinantes que la bacteria pueda expresar y transportar. En la presente revisión, se discutirán los avances más recientes en el campo de las vacunas vivas atenuadas de Salmonella, su evacuación preclínica y clínica y, también, su aplicación como vector de antígenos. Se darán a conocer las técnicas biomoleculares de clonación y expresión procariótica de las toxinas diftérica, tetánica y de pertusis, así como de los antigénos de Helicobacter pylori y de Plasmodium falciparum. Finalmente, se propone el uso de Salmonella atenuada como vector de vacunas de ADN para expresión eucariótica de los antígenos recombinanes. Los continuos esfuerzos científicos y tecnológicos en el campo de la vacunación con vectores vivos atenuados sugieren que Salmonella es una herramienta potencialmente útil para enfrentar el constante reto de la fiebre tifoidea. Igualmente, los estudios preclínicos y clínicos de fase I demuestran la eficacia de la vacuna de Salmonella viva atenuada como vector de antígenos heterólogos. Se requiere un mayor número de estudios clínicos de fase II y III para demostrar que los vectores de Salmonella podrán formar parte del arsenal de vacunas para la protección de la población mundial contra el constante acecho de los agentes infecciosos emergentes y reemergentes del presente siglo

Studies on heterologous resistance between schistosomiasis mansoni and fascioliasis

Schistosomiasis mansoni and F. gigantica GSTs were purified from adult worm homogenates by affinity chromatography. Assessment of both SmGST and FgGST as candidate vaccines was done by comparing vaccinated mice groups challenged with S. mansoni cercariae with control ones in terms of worm load, tissue egg number viability and maturity, and the survival rate of the animals. SmGST 5ug vaccination generally appeared to confer best protection against homologous challenge followed by FgGST 5 ug, while vaccination with both antigens using low doses [tug] appeared to have no specific role in decreasing worm load but had significant effects on egg production

Evaluation of intradermal sensitivity test in patients submitted to heterologous serotherapy

This review deals with heterologous sera produced used in Brazil. The authors studied 64 patients. Of these, 35 had been attacked by domestic and wild animals and received antirabies serum; 20 had been bitten by venomous animals (snakes and scorpions) and were treated with specific antivenoms; and 9 had traumas and received antitetanic serum. All these patients were submitted to the intradermal sensitivity test before, and 30 days after receiving heterologous serotherapy. The following results obtained by the authors agree with those in literature: - The intradermal test using undiluted heterologous serum produced a more acute reaction than that using heterologous serum diluted 1:10; - The larger the volume of serum, the larger was the wheal directly after inoculation; - The antigenic concentration influenced the final local reaction; - The reading 30 minutes after inoculation was always higher than that 15 min after; - No systemic reaction was observed during the tests; - The use of promethazine as a prophylactic medication did not protect against the early reactions; - Tests performed 30 days after serotherapy showed a higher reactivity, probably due to sensitization; - The intradermal sensitivity test did not allow the authors to predict early reactions. After these observations, the authors do not recommend the intradermal sensitivity test for patients submitted to heterologous serotherapy. They, however, strongly recommend a careful observation during the infusion and clinical follow up in the first 24 hours.

Response to heterologous leishmanins in cutaneous leishmaniasis in Nigeria - discovery of a new focus

A pilot study was undertaken to preliminary illustrate the leishmanin skin test (LST) positivity to distinct antigen preparations (derived from promastigote of either Leishmania major or L. amazonensis, or pooled L. mexicana, L. amazonensis and L. guyanensis) in cutaneous leishmaniasis (CL) patients and healthy subjects living in two endemic foci in Nigeria. The study was designed to provide insights into whether cross-species leishmanin, such as that prepared from New World Leishmania could be useful to detect cases of Old World leishmanial infection and to compare the results with LST using L. major-derived leishmanin. The overall LST positivity in individuals from Keana tested with the cross-major-derived leishmanin was 28.7 per cent (27/94), while the positivity rate in the subjects from Kanana tested with the same leishmanin was 54.5 per cent (6/11). Lower positivity values were obtained when L. major (12.5 per cent; 11/88) or L. amazonensis (15.8 per cent; 9/57) was tested as antigen in grossly comparable populations. Moreover, the pooled leishmanin identified most of the subjects (13/14; 92.9 per cent) with active or healed CL, and the maximum reaction sizes were found among positive subjects in this group. No healthy controls (10 total) showed specific DTH response. The LST was useful for assessing the prevalence of subclinical infection and for measuring CL transmission over time. We report for the first time the occurrence of CL in Kanana village of Langtang South local government area of Plateau State.